Stroke treatment utilizing extravascular circulation of oxygenated synthetic nutrients to treat tissue hypoxic and ischemic disorders

ABSTRACT

A novel acute care cerebral support system and method for treating severly ischemic brains is disclosed wherein an oxygenated nutrient emulsion is circulated through at least a portion of the ventriculo-subarachnoid spaces. The nutrient emulsion contains an oxygenatable non-aqueous component, an aqueous nutrient component, an emulsification component, and other components which render physiologic acceptability to the nutrient emulsion. The disclosed system and method have been shown to effectively exchange oxygen, carbon dioxide, glucose, and other metabolites in severely stroked brains. Significant restoration of oxidative metabolism and electrographic activity result from the disclosed treatment. Methods for producing the nutrient emulsion and a system for delivering that emulsion to the cerebrospinal pathway are also disclosed. Additionally, novel diagnostic methods for diagnosing the physiologic state of hypoxic-ischemic and other diseased neurologic tissue during treatment are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of Ser. No. 354,346,filed Mar. 3, 1982, which is a continuation-in-part of U.S. patentapplication Ser. No. 139,886, filed Apr. 14, 1980, now U.S. Pat. No.4,378,797, entitled "Extravascular Circulation Oxygenated SyntheticNutrients to Treat Tissue Hypoxic and Ischemic Disorders", as well asSer. No. 275,116, filed June 18, 1981, now U.S. Pat. No. 4,393,863 andSer. No. 275,117, also filed June 18, 1981, which are in turndivisionals of Ser. No. 139,886. These applications are herebyincorporated by reference as if fully set forth herein.

BACKGROUND OF THE INVENTION

Cerebrovascular accident, a disease commonly known as "stroke", remainsthe third leading cause of death, and probably constitutes the singlelargest category of long term disability in this country. In spite ofcurrent medical knowledge and available treatments, a major centralnervous system vascular occlusion is quickly attended by irreversibledamage to the affected brain region(s). A "completed stroke" is manifestby a fixed and permanent neurological deficit. Millions of dollars havebeen expended in stroke research and care by Federal and privateagencies without a single substantial gain in our presentchemotherapeutic abilities for a completed stroke.

On a clinical level, once vascular flow in any portion of the centralnervous system has ceased for longer than a few minutes, a permanent"stroke" invariably follows. It is not currently possible to recoversubstantial neural function with clinical ischemia of 5-7 minutesduration. An exquisite neuronal sensitivity to oxygen deprivation hasbeen blamed for this ultra-short stroke irreversibility. Neurons doindeed have meager metabolic storage and are unable to meet energy needsby anerobic means. Well accepted concepts hold that such permissiblecerebral ischemia times are critical and neurons must quickly beresupplied or metabolic infarction will result. While clinically true,recent laboratory investigations have addressed the problems of ischemicvascular and neuronal reactions separately with considerably differentresults. Recently reported studies indicate neurons are not as sensitiveas previously believed. Indeed, it has been suggested that neurons canwithstand global ischemia for 1 hour or longer. K. A. Hossman, P.Kleihues, Arch. Neurol. 29, 375-389 (1973). If the clinical andexperimental observations are to be reconciled, one hypothesis is thatlong-term damage results from vascular rather than neuronal sensitivityto oxygen deprivation. It is known that secondary reactive changesappear within the microcirculation after sufficient stagnation. A. AmesIII, R. L. Wright, M. Kowada, J. M. Thurston, G. Majno, Am. J. Pathol.52, 437-448, (1968). J. Chiang, M. Kowada, A. Ames III, Am. J. Pathol.52, 455-476 (1968). E. G. Fischer, Arch Neurol. 29, 361-366, (1973). E.G. Fischer, A. Ames III, E. T. Hedly-Whyte, S. O'Gorman, Stroke 8,36-39, (1977). Even if blood is represented to the local tree, the smallvessels do not completely reopen. Under these circumstances ischemic,though potentially recoverable, neurons may be lethalized because theyare not adequately resupplied with blood within their metabolicallytolerable limits. This concept shifts the basic fault in stroke from"ultrasensitive" neurons to a protracted blood flood failure.Nonetheless, a long felt need exists to prevent permanent damage and/orreverse neurologic deficits resulting from interrupted vascular flow.

One experimental approach which has been used to investigate the effectsof stroke on neurologic tissue is the perfusion of fluids of knowncomposition through ventriculocisternal spaces. For example, E.Fritschka, J. L. Ferguson and J.J. Spitzer have reported increases infree fatty acid turnover in cerebral spinal fluid during hypotension indogs. According to the Fritschka technique, a "mock" cerebral spinalfluid containing radio-labelled palmitate was perfused from the lateralventricle to the cisterna magna of conscious dogs. Arteriovenous glucoseand fatty acid concentrations, and "mock" CSF fatty acid concentrationswere monitored over a period of 6 hours of perfusion. Estimates of theamount of palmitate recovered from the cisternal effluent and cerebralvenous blood lead to the conclusion that a sizeable fraction of freefatty acids may be taken up by tissues "in the vicinity of the CSFspace". See Fritschka et al, "Increased Free Fatty Acid Turnover in CSFDuring Hypotension in Dogs", American Journal of Physiology,232:H802-H807. In "Bulk Flow and Diffusion in the Cerebral Spinal FluidSystem of the Goat", by Heise, Held, and Pappenheimer, a ventriculocisternal perfusion method was used on chronically prepared,unanaesthized goats. Measurements were made of steady-state rates atwhich inulin, fructose, creatinine, urea, potassium, sodium, andlabelled water were removed from perfusion fluid at various hydrostaticand osmotic pressures. The subject perfusions were carried out on femalegoats provided with implanted ventricular and cisternal guide tubes orcannulas. Each clearance period involved perfusion of 70-120 mills offluid through the ventricular cisternal system. Inflow rate wasmaintained constant in the range of 1.50-2.00 ml-min, and outflow wasmeasured continuously. The data obtained was used to investigate theeffects of hydrostatic pressure on inulin clearance, rate of formationof CSF, and the permeability of the ventricular system, particularly ascompared with that of the toad bladder. This ventriculo cisternalperfusion method was first reported by Pappenheimer, Heise, Jordan andDowner in "Perfusion of the Cerebral Ventricular in UnanaestheizedGoats", American Journal of Physiology, Vol. 203, pp. 763-774 (1962).Pappenheimer et al reported that goats are anatomically andtempermentally suited for ventricular cisternal perfusions and cantolerate such perfusions for many hours without showing signs ofdiscomfort. The volume of the ventricular system and rate of productionof CSF are at least double corresponding values reported for large dogs,and the thickness of the goat occipital bone and its shape facilitatesretrograde placement of cannulas through the occipital bone into orabove the cisterna magna without interfering with muscles in the neck.The goat's horns provide natural mechanical protection for the cannulasand "are almost indispensable" for operative procedures. In accordancewith the Pappenheimer et al technique, guide tubes are implanted justabove the dura over the cisterna magna and just above the ependymallinings of the lateral ventricules. Prior to each perfusion the cisternaand ventricle are punctured with sharp probe needles extending a fewmillimeters beyond the tips of the guide tubes. Alternatively, cannulaswere implanted in the subarachnoid space over the parietal cortex, thuspermitting perfusion of the entire ventriculo cisternal-subarachnoidsystem. Pappenheimer et al followed detailed protocols for implantingthe guide tubes, and for preparing sterile, synethetic CSF. ThePappenheimer et al perfusion circuit is reported to comprise a bottlesealed with a rubber cap having two stainless steel tubes extending tothe bottom of the bottle. One tube serves as a gas bubbler, the secondas a liquid outlet. A third opening connects with atmosphere through asterile cotton plug. The bottle is mounted on an indicating balance andthe reservoir outflow is connected through tubing to a parastalic pumpwith a variable drive permitting pumping rates in the range of 0.5-5ml/min. One pump output is lead to a male syringe joint which fits theventricular probe needles and a second outlet on the joints connects toa strain gauge manometer. A 5 ml empty sterile syringe is placed inparallel with the output to damp pulsations of the pump. The cisternaloutflow is connected to an enclosed drop counter and wing flask and theoutput is recorded cumulatively on a polygraph which also gives avertical record proportional to outflow rate. Pappenheimer et al reportsthat perfusion with CSF of normal composition can usually be maintainedfor 4-8 hours before the animal becomes resistive, and if correctlyperformed, the animal will show no sign of knowing when the perfusionpump is on or off. No attempt is made to regulate the temperature offluid entering the ventricular probe, however at flow rates of 1-2ml/min it is theorized that the fluid reaches temperature equilibriumwith the brain before reaching the hypothalmus. At higher flow rates(4-6 ml/min) the animals are reported to start to shiver. In thisregard, see also F. H. Sklar and D. M. Long, Neurosurgery 1, 48-56(1977).

Over the years, many experiments have been conducted with materialspossessing high oxygen-dissolving properties, many of which have beenincorporated as constituents in "artificial blood". The concept ofutilizing materials possessing high oxygen-dissolving properties for themaintenance of tissue respiration was first reported by Rodnight in1954. See Rodnight, R., Biochemistry Journal, Vol. 57, p. 661. Rodnightcapitalized upon the considerable oxygen solubility found in siliconeoils, and sustained tissue slices by incubation in these oxygen ladenoils. Approximately 12 years later, Clark reported experiments involvingthe total immersion of small animals in silicone oils and fluorocarbonliquids. Rats totally immersed in oxygenated silicone oil survived forone hour with no apparent ill effects, but died several hours afterremoval, from unknown causes. Similar experiments using syntheticfluorocarbon liquids, which dissolve about 3 times more oxygen than dothe silicone oils, were performed with some success. Under theseconditions animals survived immersion in oxygenated syntheticfluorocarbon liquids and thereafter returned to apparent health. SeeClark, L. C. Jr. and Gollon F., Science, Vol. 152, p. 1755, (1966); andGollon, F., Clark, L. C. Jr., Alabama Journal of Medical Science, Vol.4, p. 336, (1967). While arterial oxygenation was reported as excellentfor Clark's studies in rats, coincident impairment of carbon dioxideelimination was also reported, as was pulmonary damage from breathingfluorocarbon liquids. One rat, which was observed for five daysfollowing liquid breathing, was described as being in respiratorydistress and as succumbing within 15 minutes after the subcutaneousadministration of hydrocortisone (50 mg), with copious loss of bodyfluid from the trachea. In this regard, Clark concluded:

These organic liquids should prove to be of value in studies of gasexchange in living tissues in animals. Organic liquids, since they cansupport respiration with oxygen at atmospheric pressure and have otherunique qualities, may find use in submarine escape, undersea oxygensupport facilities, and medical application. The pulmonary damage causedby the breathing of the organic liquids available at the present timeremains a major complication of their use in man. Science, Vol. 152., p.1756.

See also K. K. Tremper, R. Lapin and E. Levine, Critical Care Medicine8:738 (1980); S. A. Gould, A. L. Rosen, L. R. Sehgal, Fed. Proc. 40:2038(1981).

Following these observations, fluorocarbon liquids were used as anincubation medium for isolated rat hearts. See Gollon and Clark, ThePhysiologist, Vol. 9, p. 191, (1966). In this work, myocardial oxygenrequirements were apparently well met, however these hearts did notflourish without intermittent fluorocarbon removal and washing withoxygenated, diluted blood. This phenomenon has been explained in termsof aqueous phase lack in pure fluorocarbons such that necessary ionicexchange is impeded.

More recently, considerable attention has been directed to the use offluorocarbons as constituents of artificial blood. Sloviter, in order toovercome the problem of aqueous-metabolite fluorocarbon insolubility,made an emulsion with fluorocarbon and albumin. Sloviter's emulsionsustained the isolated rat brain by a vascular perfusion as well as didan erythrocyte suspension. See Sloviter, H. A. and Kamimoto T., Nature(London), Vol. 216, p. 458 (1967). A better emulsion was later developedcomprising a detergent, "Pluronic F 68" (manufactured by the WyandotteChemical Corp., Wyandotte, Mich.), and fluorocarbon liquids which wereproperly emulsified using sonic energy. This improved emulsion permittedthe replacement of most of the blood of a rat which was then reported assurviving in an atmosphere of oxygen for five to six hours. See Geyer"Survival of Rats Totally Perfused with a Fluorocarbon-DetergentPreparation", Organ Perfusion and Preservation, edited by V. C. Normen,N.Y.: Appelton-Century-Crofts, pp. 85-96 (1968), Geyer, R. P.,Federation Proceedings, Vol. 29, No. 5, September-October, 1970; andGeyer, R. P. Med u Ernohn, Vol. 11, p. 256 (1970).

Experiments have also been reported wherein fluorocarbons have been usedto perfuse livers. Ten hours after in vitro fluorocarbon perfusion, theisolated liver ATP; AMP; lactate/pyruvate ratio; and a number of othermetabolites were found to be as good or better than livers perfused invitro with whole blood. See Krone W., Huttner, W. B., Kampf S. C. etal., Biochemika et Biophysica Acta, Vol. 372, pp. 55-71 (1974). Thesedetailed metabolic studies indicated that the organs perfused with 100%fluorocarbon liquid were redeemed "intact"; while only 75% of the wholeblood infused organs maintained a similar degree of metabolic integrity.The ability of fluorocarbon perfusion to maintain cellular integrity wasconfirmed by electron-microscopy studies. The cells had normalmitochondrial ultra structure after ten hours of fluorocarbon support,indicating the persistence of normal or adequate aerobic metabolism. InBrown and Hardison, "Fluorocarbon Sonicated as a Substitute forErythocytes in Rat Liver Perfusion", Surgery 71, pp. 388-394 (1972) afluorocarbon perfusate preserved organ function and integrity far betterthan perfusate with much lower oxygen carrying capacity, but wasreported as resulting in a decreased rate of bile secretion which wasprobably the earliest sign of hepatic damage, tissue edema, and areproducible rise of portal pressure over a period of 21/2 to 3 hours.Both tissue edema and rising portal pressure with fluorocarbon perfusionwere associated with progressive vascular occlusion as determinedhistogolically. A greatly diminished perfusion of fluorocarbon at theend of experiments was documented by injection of India ink twentyminutes before the end of the perfusion. Brown and Hardison hypothesizedthat the fluorocarbon perfusate may react with amino acids and proteins,that the oxygen concentration in the fluorocarbon perfusate may affectthe perfusion results, and that filtration of the fluorocarbon emulsionthrough filter paper and differing instrumentation were responsible forthe apparently conflicting results in the literature. Brown and Hardisonhypothesize that phagocytosis of fluorocarbon particles might completelyblock reticticuloendotheilial cells in liver or that capillaryendotheilial damage may be another reason for late fluorocarbonperfusion problems.

Fluorocarbons have also been used in experiments involving cerebralblood circulation. In Rosenblum's studies, mouse hematocrits werereduced to 10-15 by exchanging the animal's blood with a fluorocarbonsolution. When the animals were respired with 100% oxygen afterintravascular fluorocarbon infusions, the brains remained metabolicallysound. These organs were able to reverse rising NADH levels and EEGabnormalities induced by short period nitrogen inhalation. The EEG's offluorocarbon treated animals could be activated by the central nervoussystem stimulant metrazole. By these criteria, intravascularfluorocarbon does support the cerebral microcirculation and providesfunctions of oxygenation, metabolism and electrical activity which arenormally associated with blood transport. Please refer to Rosenblum, W.I., "Fluorocarbon Emulsions and Cerebral Microcirculation", FederationProceedings, Vol. 34, No. 6, p. 1493 (May 1975). See also S. J.Peerless, R. Ishikawa, I. G. Hunter, and M. J. Peerless, Stroke 12, pp.558-563 (1981); B. Dirk, J. Creiglstein, H. H. Lind, H. Reiger, H.Schultz, J. of Pharm. Method 4, pp. 95-108 (1980); J. Suzuki, T. Y.Oshomoto, S. Tanaka, K. Moizoi, S. Kagawa, Current Topics 9, pp. 465-470(1981).

As reported by Kontos et al, the marked vasodilation of small cerebralsurface arteries which occurs in response to acute profound hypoxemiamay be locally obviated by perfusing oxygen equilibrated fluorocarboninto the space under the cranial window. See Kontos, H. A., et al, "Roleof Tissue Hypoxemia in Local Regulation of Cerebral Microcirculation",American Journal of Physiology, Vol. 363, pp. 582-591 (1978). Kontos etal described the effect of perfusions with fluorocarbon with 100% oxygenas resulting from increased supplies of oxygen to the neural cells andconsequent partial or complete relief of hypoxia, rather than to a localincrease in the oxygen tension in the immediate environment of thevascular smooth muscle of the pial arterioles. Two other potentialexplanations for the observed action are also suggested in the Kontos etal article.

In 1977, Doss, Kaufman and Bicher reported an experiment wherein afluorocarbon emulsion was used to partially replace cerebrospinal fluid,with the intention of evaluating its protective effect against acuteanoxia. Doss et al, Microvascular Research 13, pp. 253-260 (1977).According to this experiment, systemic hypoxia was produced through oneminute of 100% nitrogen inhalation. A bolus of oxygenated fluorocarbonplaced in the cisterna magna immediately prior to nitrogen breathingincreased regional cerebrospinal fluid O₂ tension by a factor of 5.During the one minute experimental period, the fluorocarbon emulsionprovided twice as much brain tissue oxygen as was found in salineinjected controls. Doss et al found the anticipated regional tissueoxygenation decline attending nitrogen inhalation to be halved by theadministration of the oxygen bearing fluorocarbon emulsion.

In spite of the above described experiments, there is yet to be reportedany practical therapeutic approach to the treatment of ischemicneurologic tissue, and particularly human ischemic central nervoussystem tissue resulting from stroke, accident or disease.

SUMMARY OF THE INVENTION

The present invention provides a novel nutrient formulation forcirculation through cerebrospinal fluid pathways, and systems andmethods for using same, to treat central nervous tissue hypoxic-ischemicconditions. Through its use, a new diagnostic methodology is alsodisclosed.

Applicant has recognized that there is a therapeutic time window throughwhich neuron can be reached and resuscitated. The method of the presentinvention is designed to bypass obstructed vascular circulation anddeliver cerebral metabolic needs through an alternate cerebral spinalfluid (CSF) circulation portal. Since particle size exerts a majorinfluence on brain penetration from CSF, the method of the presentinvention is hypothesized to permit diffusion of oxygen, glucose,electrolytes and essential amino acids into ischemic neural tissue whenpresented in abundance in the cerebral spinal pathway. Thus, a rapidlyexchanging cerebral spinal fluid perfusion system is provided to amplysupply these materials and, at the same time, remove metabolic waste.

The cerebrospinal fluid (CSF) pathway system, which intimately bathesand permeates brain and spinal cord tissues, constitutes a uniqueanatomical relationship within the body. Although it has somesimilarities to systemic lymphatics, its anatomical arrangement differsconsiderably from that of lymph. Indeed, this system has been named the"third circulation". Due to the extensive area of CSF-tissue contactover the cerebral and cord surfaces, in the miniature Virchow-Robinsspaces, and cerebral ventricles, the cerebrospinal fluid systemconstitutes a vast, complex and intimate therapeutic avenue for accessto central nervous tissue. Excepting certain infections and neoplasmswhere the cerebrospinal fluid is now utilized as a treatment conduit,the cerebrospinal fluid system has not been otherwise widely exploitedas an easily accessible therapeutic route and has never been used as acontinuous therapeutic diagnostic circulation system in man. The presentinvention is predicated on the recognition that, when regional cerebralblood flow is interrupted, such as after major stroke, or is otherwiseseriously impeded by profound vaso-spastic states, the cerebrospinalfluid pathway actually represents the only practical and viableanatomical route by which these tissues may be readily treated. Thisresults from the fact that the usual vascular delivery system is eitheroccluded or non-functional, and thus tissues within affected territoriescannot be properly served.

In accordance with the present invention, essential cellular substratesare delivered to beleaguered ischemic brain regions by utilizing the"back door" cerebrospinal fluid delivery route. Accordingly, the presentinvention provides a novel nutrient emulsion, circulatory method andsystem which provide necessary nutrient penetration into regionssuffering vascular deprivation.

It has been found that the cerebrospinal fluid to brain relationship isnot characterized by the rigid and highly selective barrier mechanismwhich are present at the blood-brain interface. Thus, the penetrationrate of materials from cerebrospinal fluid regions to the brain relatelargely to molecular size, that is, small substances penetrate deeplywhile large molecules move more slowly into brain substance. Althoughentry rates are generally inversely proportional to molecular weight,penetration is also influenced by lipid solubility and the molecularconfiguration of the penetrating substance. Accordingly, the presentinvention provides a nutrient emulsion containing essential brainnutrients including selected electrolytes, having a relatively lowmolecular size which, in accordance with the methods of the presentinvention, are caused to relatively freely diffuse from either theventricular or subarachnoid fluid regions into the brain matter to betreated. Accordingly, the present invention provides a novel nutrientemulsion which has been purified, balanced, and perfected to fall withinnarrow physiologic limits while nonetheless providing the desirednutritional characteristics referred to above.

In accordance with the preferred embodiment of the present invention,this nutrient emulsion constitutes "synthetic cerebrospinal fluid"comprising preselected electrolytes, glucose, amino acids, at least oneoxygen-carrying component, typically a fluorocarbon, and othercomponents which impart to the composition a preselected pH, bufferingcapability, and osmolarity. This nutrient emulsion is prepared bycontrolling sonication time and by properly dialyzing the materials toachieve a toxic free emulsion. The resulting solution may be rapidlyoxydated to O₂ pressures of 650 mm of mercury by using the hereindisclosed modified recirculating pediatric oxygenator. As a result, anovel oxygenated nutrient emulsion is provided which is believed toexhibit exceptional therapeutic properties.

The present invention also provides a novel method and apparatus forcirculating the oxygenated nutrient emulsion through cerebrospinal fluidpathways, particularly those pathways which contact brain and spinalcord tissue. According to these methods, treated tissues exhibit asubstantially improved ability to resist and/or repair damage whichwould otherwise result from vascular occlusion. In accordance with thepreferred method of the present invention, the novel oxygenated nutrientemulsion is circulated through this cerebrospinal fluid route byinjecting it into brain ventricles and withdrawing it from the cisternamagna or the spinal subarachnoid space to nourish and to treat centralnervous tissues. In other instances the fluid may be injected into thesubarachnoid space and withdrawn from another subarachnoid position. Thepreferred embodiment oxygenated nutrient emulsion should be circulatedto tissues to be treated in amounts sufficient to provide adequate gasexchange. Pure fluorocarbon may contain 50 ml O₂ per 100 ml at oneatmosphere oxygen while normal blood contains only 20 ml O₂ /100 mlunder the same conditions. The oxygen carrying capability per ml of thefinal emulsion is considerably less than that of pure fluorocarbon byreason of its content of other constituents for normalizing osmoticpressure, buffering, electrolytes, and other physiologic balancingmaterials. Thus, the preferred embodiment nutrient emulsion may becharged with oxygen (100% O₂ at one atmosphere) to attain pO₂ tensionsof 640-700 mm of mercury and an O₂ content of 20 ml per 100 ml. Underrapid circulation conditions, the integral O₂ exchange (fluorocarbon totissue) has been found to be about 33%. Thus, an oxygen exchange valueof about 6.6 ml O₂ /100 ml nutrient emulsion per minute is provided bythe present method.

In accordance with the preferred embodiment of the present invention,sufficient nutrient emulsion should be supplied to counteract oxygendeprivation to the affected tissue. For example, the entiresupertentorial adult cat brain weighs 12 grams (±2) and the normalmetabolic consumption of oxygen of mammalian brain tissue equals 3-4 mlper 100 grams per minute. This total metabolic need may be met with thecirculation rate of 6-8 mls per minute. Metabolic needs necessary tosimply sustain and/or salvage tissue may be achieved by perfusion ratesof one half or less of this optimum. Within these constraints an easilyachieved sustenance flow rate of at least 20-30 ml/minute, optimally45-60 ml/minute, would be anticipated to salvage 100 gms of human braintissue. It has been found experimentally that it is possible to supplysufficient oxygen to counteract the deprivation of the affected tissuethrough circulation of the nutrient emulsion through the cerebrospinalfluid route. In fact, under carefully controlled conditions, it isbelieved within the scope of the present invention to nourish the entirehuman brain using the preferred embodiment apparatus, method andsubstance of the present invention. In this manner, central nervousneurons deprived of major blood supply may be sustained withoutsignificant damage.

In accordance with the preferred embodiment of the present invention, anovel system is disclosed for administering and maintaining theoxygenated nutrient emulsion for delivery and circulation through thecerebrospinal route.

The preferred embodiment system of the present invention effectivelycarries out the circulation and equilibration of the nutrient emulsionduring treatment. This system, which is diagrammatically illustrated inFIG. 1, generally comprises a reservoir containing nutrient emulsion;means for delivering the nutrient emulsion at preselected flow rates; anoxygenation means for equilibrating the nutrient emulsion to desiredgaseous tension levels; heat exchanger and/or cooling unit means forselectively controlling the temperature of the nutrient emulsion;filtering means for cleansing the nutrient emulsion; and circulationmonitoring means for insuring that desired circulation flows andpressures are maintained within the system.

The present invention also provides a method of diagnosing conditions ofneurologic tissue in mammals. This novel method generally comprisesproviding an artificial spinal fluid of known composition, injectingthat artificial spinal fluid into at least a first portion of thecerebrospinal pathway of a mammal, withdrawing a diagnostic fluid from asecond portion of that pathway to create a circulation of fluid at leastthrough a portion of said pathway, monitoring the composition of saiddiagnostic fluid, and comparing for at least a selected difference inthe compositions of said artificial spinal and diagnostic fluids,whereby the detected differences in those compositions are at leastdiagnostic of neurologic tissue disposed along said portion of thecerebrospinal pathway. In accordance with the diagnostic methods of thepresent invention, the diagnostic fluids may be monitored fordifferences in oxygen content, lactic acid concentration, carbon dioxideconcentration, potassium and/or sodium ion concentration, enzymeconcentration, pH difference, ammonium concentrations, GABA(gamma-aminobutyric acid) and other amino acid(s) concentrations,microorganism content, bacterial count, myelin fragments, cellularfragments or organelles, malignant cells, and/or poisons.

It is also within the scope of the present invention to provide a novelnutrient liquid and/or diagnostic liquid for treating cerebrospinaltissue containing various novel specified components which is formulatedusing novel methodology.

It is additionally within the scope of the present invention to providea novel apparatus for treating patients having ischemic-hypoxic tissues,including novel injection and withdrawal means comprising a novelcatheter means which is particularly adapted for injecting oxygenatednutrient liquid into a cerebral ventricle without danger ofsubstantially damaging neurologic tissue in the vicinity of thatventricle.

In addition to the methods described above, it is within the scope ofthe present invention to provide additional therapeutic agents to thenutrient emulsion, such as antineoplastic agents; antibiotics, and/orother therapeutic agents for use in treating the target tissue(s).

Accordingly, the primary object of the present is the provision of amethod, substance, and system for providing early stroke treatment.

Other objects of the present invention are to provide treatments forbrain and spinal cord injuries, cerebral hemorrhage, cerebral vasospasm,senility, after general hypoxia and other hypoxic-ischemic relatedneurological disorders.

It is a further object of the present invention to provide therapeutictreatment which may sustain the life of the brain and central nervoussystem tissues in case of profound shock and/or temporarycardio-respiratory failure.

It is a further object of the present invention to providelife-sustaining support to the brain and/or spinal cord tissues duringthe conduct of neurological or cardiovascular surgery.

Other objects of the present invention are the provision of methodswhich may compliment treatments of central nervous system neoplasms byeither external radiation and chemotherapy by providing local tissuehyperoxygenation or drugs which may enhance drug or radiationtumorocidal effects.

Further objects of the present invention include the provision ofmethods which are useful in treating anoxic states attending birthinjury. The present method will also assist in removal of centralnervous system poisons.

These and other objects of the present invention will become apparentfrom the following more detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic view of the preferred embodiment treatmentsystem of the present invention illustrating the circulation of nutrientemulsion from a reservoir, into a cerebral ventricle, such as a lateralventricle, through a portion of the cerebrospinal fluid pathway foroutput from the spinal subarachnoid space or from the cisterna magna;

FIG. 2 is a diagrammatic view of a portion of the preferred embodimenttreatment system of FIG. 1 illustrating an alternate circulation routewherein oxygenated nutrient emulsion is injected into the spinalsubarachnoid space and is collected from the cisterna magna;

FIG. 3 is a diagrammatic view of portion of the preferred embodimenttreatment system illustrated in FIG. 1 showing an alternate circulationroute wherein oxygenated nutrient emulsion is injected into the cisternamagna for passage through the spinal subarachnoid space for withdrawalfrom a lumbar region;

FIG. 4 is an EEG power recording from the left and right hemispheres ofa cat showing traces from the time of an initial stroke, at the end ofthe stroke, and four hours after the stroke;

FIG. 5 is an EEG recording of an animal perfused with oxygenatednutrient emulsion having a pO₂ level of 400 and showing a 5% return ofEEG at 4 hours;

FIG. 6 is an EEG similar to FIG. 1 for an animal perfused withoxygenated nutrient emulsion having a pO₂ of 645 and showing an 88%return of electrocerebral power within 4 hours;

FIG. 7 is an EEG trace showing the effect on EEG activity of a temporarycessation in oxygenated nutrient emulsion circulation;

FIG. 8 is a graph showing the effect on glucose metabolism (CMRGl),lactate and pyruvate before and after stroke of a perfused animalparticularly illustrating the effect of a reduction in perfusion rate toinsubstantial levels;

FIG. 9 is a bar graph showing the mean EEG recovery (percent) for groupsof cats subjected to strokes resulting in 15 minutes of EEGisoelectricity, and comparing naive animals to those perfused only withartificial cerebral spinal fluid (lumbar) and oxygenated nutrientemulsion through lumbar and cisternal routes;

FIG. 10 is a graph of microequivalents of potassium per minute versustime for two experimental groups of cats subjected to 15 minutes of astroke induced isoelectric state;

FIG. 11 is a graph similar to FIG. 10 wherein the data in FIG. 10 isrepresented as a percent of the base line figure;

FIG. 12 is a glucose metabolism (CMRGl) graph plotting milligrams pergrams per minute against time for three perfusions using a standardglucose concentration, one perfusion using twice that glucoseconcentration, and a control using artificial cerebral spinal fluidwithout fluorocarbon;

FIG. 13 is a diagrammatic view of an alternate embodiment oxygenatednutrient emulsion delivery system for use in performing the methods ofthe present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following more detailed description, numerous examples have beenselected for the purposes of explanation and illustration of thepreferred embodiments of the present invention. One of ordinary skill inthe art will recognize that various changes may be made in the materialsand methods disclosed herein without departing from the scope of thepresent invention, which is defined more particularly in the appendedclaims.

Referring now to FIG. 1, the preferred system for circulating nutrientemulsion through a cerebrospinal pathway is diagrammaticallyillustrated. As shown in FIG. 1, a nutrient emulsion reservoir 10 isprovided for receiving and retaining nutrient emulsion, the preparationof which will be described more fully hereinafter. In accordance withthe preferred system and method of the present invention, the nutrientemulsion is injected into a cerebrospinal pathway following pHadjustment and filtering, temperature adjustment, oxygenation, andadjustment of the pressure and flow rate of the nutrient input stream.In FIG. 1, these steps are illustrated diagrammatically at 12, 14, 16and 18 respectively. Preferably, the nutrient input stream is deliveredto a ventricle of the brain, and more particular to a lateral ventricle20 of the human brain, designated generally 22. Injection of thenutrient input stream permits the oxygenated nutrient emulsion to comeinto contact with the subarachnoid spaces, miniature Virchow-Robinsspaces, cerebral and cord surfaces, and cerebral ventricles. For thesystem illustrated in FIG. 1, the nutrient input stream isdiagrammatically illustrated as being injected into a lateral ventricle20. Since the lateral ventricle is in fluid communication with otherportions of the cerebrospinal pathway, withdrawal of fluid from aportion of the pathway which is remote from that ventricle will create acirculation of fluid within the cerebrospinal pathway. More particularlycirculation of the nutrient input stream though at least a portion ofthe cerebrospinal pathway may be accomplished by withdrawing fluid fromthe spinal subarachnoid space, diagrammatically illustrated as 26 inFIG. 1, or alternatively, from the cisterna magna 24.

It is not necessary to conduct steps 10-18 in the sequence illustratedin FIG. 1. In FIG. 13 the presently preferred apparatus for deliveringoxygenated nutrient emulsion is diagrammatically illustrated. Thisapparatus, which may be easily constructed using a pediatric bloodoxygenator such as an H-800 Pediatric Oxygenator available from TheWilliam Harvey Cardiopulmonary Division of C. R. Bard, Inc., Santa Ana,Calif. 92705, comprises a nutrient emulsion reservoir having oxygenationand temperature adjustment loops for constantly oxygenating andadjusting the temperature of the nutrient emulsion contained within thereservoir. In this manner the flow rates of nutrient emulsion providedfrom oxygenation by oxygenator 102 or for temperature adjustment 104 maybe independently varied through adjusting the flow rate of delivery byvariable speed pumps 103 or 105 to optimize the temperature and pO₂characteristics of the oxygenated nutrient emulsion to be delivered forinjection by variable speed delivery pumps 106 and 107. As normallyused, pediatric blood oxygenators fail to provide a sufficient oxygentransfer rate to fluid flow rate to accommodate the emulsion of thepresent invention. The minimum blood flow rate of the H-800 oxygenator,for example, is 0.5 liters per minute, and the oxygen transfer rate (toblood) at this flow rate is less than about 25 ml/min. By routing theoutput 101 of the oxygenator to the reservoir, the oxygenator outputpump 103 may operate at flow rates which easily achieve about 7 litersper minute of oxygen transfer to the fluorocarbon carbon emulsioncontained in the 2000 ml reservoir. At the same time, delivery pumps 106and 107 may provide much lower flow rates of nutrient emulsion to theanimal undergoing treatment. In a similar manner, heat exchange may alsobe optimized. In order to maintain optimal pO₂ values, each conduit ofthis system should be composed of an oxygen impermeable material toprevent leakage of oxygen from the oxygenated nutrient emulsion duringprocessing and delivery. The filtration and chemical balancingprocedures followed in preparing the nutrient emulsion are not presentlyperformed "on line", however it is anticipated that chemical balancingmay be performed as a closed loop process, as illustrated in FIG. 13.Filtration 108 is performed on line under pressure from pump 106 using amillipore bacterial filter. Pump 107 establishes the final injectionrate. The flow of nutrient emulsion to the chemical balancing system isadjusted using variable speed pump 111. In the embodiment of FIG. 13,pressure monitoring and control is accomplished using an open side arm114 bearing indicia thereon which correspond to the hydraulic pressureof oxygenated nutrient emulsion within delivery line 19. The height ofthe side arm is adjusted so that overflow will occur when the maximumdesired intracranial pressure has been obtained.

As shown in FIG. 1 the oxygenated nutrient emulsion input stream iscarried through input stream conduit 19 to an injection cannula 20awhich is coupled thereto by coupling 21. Injection cannula 20a isrigidly attached to skull 22 by fitting 22a which holds the cannula inits proper orientation to permit injection of the oxygenated nutrientemulsion into lateral ventricle 20.

If preferred, a double lumen catheter, such as catheter 120 (FIG. 13),may be utilized in place of input cannula 20a. One of the lumens of thiscatheter should be connected to a pressure monitoring means formonitoring the intracranial pressure within the lateral ventricle 20.This pressure monitoring means may comprise an open side arm, such asside arm 122 which functions similarly to side arm 114.

The preferred injection means of the present invention comprises acerebral catheter means for insertion into a brain ventricle. Thisinjection means comprises means for preventing a portion of the catheterlocated within a brain ventricle from damaging tissues surrounding theventricle. In the preferred embodiment, an inflatable balloon tip may beprovided for this purpose. The actual injection of nutrient emulsioninto the brain ventricle is accomplished by providing an arrangement ofoutlet holes disposed as a series of slits radially spaced around thecatheter tip. Both the injection means and withdrawal means also furthercomprise attachment means for attaching the catheter to the body in thevicinity of the injection or withdrawal sites. Thus the injectioncatheter may comprise a means for fixing at least a portion thereof withrespect to the skull to insure catheter stability. The withdrawalcatheter, which may have a tip with multiple perforations disposedtherein, further comprises means for attaching at least a portionthereof to tissue in the region of the subarachnoid space. Thisattachment means may include a staple for attaching a non-collapsibleportion of the catheter to a lumbar region of the skin.

In many applications, the oxygenated nutrient emulsion will be deliveredunder normothermic conditions, that is, at about 37° C. Under theseconditions, and under hypothermic or hyperthermic conditions where thedelivery temperature of oxygenated nutrient emulsion is higher thanambient temperature, temperature adjustment is easily accomplished byproviding a thermostatically controlled heater coupled to a suitableheat exchanger for adjusting the temperature of oxygenated nutrientemulsion recirculated to the nutrient emulsion reservoir.

The circulation route illustrated in FIG. 1 permits the treatment of atleast cerebral tissues. It is within the scope of the present invention,however, to focus treatment on selected neural tissue areas, in whichcase alternative points of injection and withdrawal of fluid may beselected by the attending physician. For example, in the case of spinalcord injury, it is anticipated that the point of injection of oxygenatednutrient emulsion may be the lumbar, spinal subarachnoid space, with thepoint of withdrawal being the cisterna magna. While the above mentionedcerebrospinal pathway injection and withdrawal points are preferred, itis within the scope of the present invention to utilize other injectionand withdrawal locations, provided a substantial circulation of fluidthrough the area of affected neurologic tissue is established byutilizing the selected loci. Such alternate pathways are illustrated inFIGS. 1-3. In FIG. 1, withdrawal of the nutrient emulsion from thecisterna magna is illustrated via conduit 30 in dotted outline. In FIG.2 input conduit 19 injects oxygenated nutrient emulsion into thediagrammatically illustrated subarachnoid space 26. Withdrawal from thecisterna magna is via conduit 30b. In FIG. 3 injection into the cisternamagna is accomplished via injection catheter 30a. Withdrawal is from thediagrammatically illustrated spinal subarachnoid space 26 via withdrawalcatheter 30c.

The fluid which is withdrawn from the cerebrospinal pathway will not beof identical composition to the oxygenated nutrient emulsion which isinjected at the injection point. By taking advantage of differences incomposition which are detected in the withdrawn fluid, which may beconsidered to be a diagnostic fluid, the attending physician may easilymonitor the physiologic condition of the neurologic tissue which isbeing treated. This diagnostic fluid may also be monitored to assurethat treatment is proceeding according to plan. Accordingly, fluid whichis withdrawn from the cerebrospinal pathway is directed to an outputcollection means 28 for collecting diagnostic fluid. Preferably, anoutput monitor 34 will continuously monitor various chemical andphysical characteristics of the diagnostic fluid for such properties asflow rate, hydraulic pressure, potassium and sodium ion concentration,temperature, lactic acid concentration, gamma amino butyric acid andother amino acid concentrations, oxygen concentration, carbon dioxideconcentration, enzymes, and ammonia concentration. The output of thisoutput monitor will not only provide the attending physician withinformation concerning the state of the cerebrospinal tissue beingtreated, but also will be fed back to the monitor, control and alarmsystems for at least pressure and flow rate, temperature, oxygen-carbondioxide and chemical constituency, as described more fully hereinafter.This diagnostic system takes advantage of the fact that ischemicneurologic tissue produces higher concentrations of such materials asGamma-aminobutyric acid (GABA), lactate ion (lactic acid), enzymesand/or LDH (lactic dehydrogenase), ammonia, and other constituents whichhave been determined by analyzing cerebrospinal fluid of patientssubjected by disease to similar anoxic conditions.* In accordance withthe system of the present invention, however, a continuous monitoring ofthe state of neurologic tissue is possible, since the circulation ofoxygenated nutrient emulsion will produce a continuous flushing of theaffected tissue regions, and thus will result in diagnostic fluidcomponent variations which are rapidly reflective of the physiologicstate of the tissues being treated. Due to the multipointinjection-withdrawal method of the present invention, dangers which areinherent in sampling natural cerebrospinal fluid at a single locationare avoided by utilizing a double venting method wherein thecerebrospinal fluid pressure is at all times carefully controlled.

It is within the scope of the present invention to sterilize andreconstitute that diagnostic fluid as shown at step 32, whereupon thatreconstituted diagnostic fluid may be provided as nutrient emulsion tothe nutrient emulsion reservoir 10. As shown in FIG. 1, the outputmonitor 34 may monitor the diagnostic fluid during the sterilization andreconstitution processes and, if desired, ensure that the reconstitutedfluid satisfies the requirements of the nutrient emulsion reservoir. Asshown in FIG. 1, in order to ensure that appropriate degrees ofoxygenation, filtration and chemical balancing, temperature adjustment,and pressure and flow rate are maintained, the nutrient input stream ismonitored by various monitors, controls, and alarms, which are intendedto provide a fail-safe nutrient input stream. In particular, a pressureand flow rate monitor, control and alarm 38 is provided for monitoringthe pressure and flow rate of the nutrient input stream, for controllingthe pressure and flow rate adjustment 18 to establish desired pressuresand flow rates, and for sounding an alarm in the event that the nutrientinput stream exceeds or falls below preselected pressures or flow rates.If desired, this alarm may additionally disable the pumping mechanismproducing flow of the nutrient input stream such that the unit "shutsdown" upon detection of unacceptable input stream conditions.

Referring now to the temperature monitor, control and alarm, thetemperature characteristics of the nutrient input stream are similarlydetected, at least to ensure that hyperthermic states, except when usedas therapeutic modality, are avoided. While in most instances, thenutrient input stream will be adjusted to a 37° C. temperature, it maybe desired to select hypothermic temperatures in order to establishcertain treatment conditions. In either event, the temperature monitorwill continuously detect the temperature of the input stream, willcontrol the temperature adjustment 14 to establish a preselectedtemperature, and will sound an alarm and/or disable the system in theevent that a preselected temperature range is not maintained in thenutrient input stream.

Referring now to the chemical monitor, control and alarm 42, thenutrient input stream will be continuously monitored for one or morechemical or physical characteristics of the nutrient input stream, andwill control the chemical balancing, filtration, etc. which is performedby the filtration and chemical balancing unit 12. The chemical monitor,control and alarm may, for example, monitor the pH, osmolarity,electrolyte component, carbohydrate component, amino acid component, orother components of the nutrient emulsion to ensure that the nutrientinput stream falls within preselected stream characteristics. In theevent that these characteristics do not fall within the preselectedrange, the alarm for unit 42 may sound and/or may disable the system tothereby prevent further injection of nutrient input stream into thecerebrospinal pathway.

Finally, an oxygen/carbon dioxide monitor, control and alarm unit 36 isprovided which continuously monitors the oxygen and carbon dioxidecontents of the nutrient input stream, which controls the oxygenationunit 16, and which sounds an alarm in the event that the oxygen orcarbon dioxide concentrations do not fall within preselected ranges. Itis anticipated that each of units 36-42 may provide continuous displaysof the information monitored from the nutrient input stream, and may, ifdesired, enable back-up units which either manually or automaticallysupplement or replace the functions of units 12-18 in the event thatthose units are not functioning to produce a nutrient input streamwithin the desired ranges. For example, it is anticipated that a manualor battery operated pump, oxygenator, filter, and pressure and flow rateadjustments be provided to enable emergency operation of the system,since continual nutrient flow is lifesaving for the devitalized portionof the treated organ.

The preferred nutrient emulsion of the present invention is comprised ofcarefully formulated components which, to the extent possible whilemaintaining desired therapeutic activity, mimic the physical andchemical characteristics of natural cerebrospinal fluid. Generally,tissues and cells will not fair well if exposed to large volumes ofnon-physiologic ionic solutions. Accordingly, it has been recognizedthat appropriate electrolyte compositions at the tissue level areindispensable when it is considered that the circulatory method of thepresent invention would otherwise result in the washing and the dilutionof electrolytes from the region even after short terms of circulation,to the detriment of cell membrane functions. Accordingly, in accordancewith the preferred embodiment of the present invention, sodium,potassium, calcium, magnesium, and chloride ions are carefully balancedin the nutrient emulsion of the present invention to thereby create, tothe degree possible, normal extra-cellular compositions. The presentinvention also provides a non-aqueous oxygen transfer component forselectively combining with oxygen and for transferring oxygen to thetissues to be treated. Numerous compounds are known to the art which arecharacterized by having a high solvent property for oxygen, carbondioxide, and other gases. The preferred non-aqueous oxygen transfercomponent of the preferred nutrient liquid should exhibit when socharged, oxygen vapor pressure ranges of above 400, and preferably 600,Torr. Such oxygen transfer components should similarly not have inthemselves high vapor pressures which would boil at body temperatures,nor have viscosities which are difficult if not impossible to emulsify.Generally, the preferred compounds for use as non-aqueous oxygentransfer components are fluorocarbon polymers, such as perfluorocarbons,perfluorinated alkyl polyethers, fluoroethers, fluoramines, etc. Whilecompounds within these groups range in molecular weight from 250 to7000, their selection for use as non-aqueous transport components arebased upon the combination of features of the proper vapor pressure,molecular weight, viscosity, and emulsifiability, emulsion-stability andtissue distribution. One such fluorocarbon which has been found to beparticularly suited for the non-aqueous oxygen transport component ofthe preferred nutrient liquid is a reagent gradeperfluorobutyltetrahydrofuran which has been sold by the 3-M Corporationunder the trademark "FC-80". FC-80 has an oxygen solubility coefficientScO₂ of 0.45 of ml O₂ /ml at pO₂ of 760 Torr. See Navari et al., Res.Exp. Med. 170, pp. 169-180 (1977), which paper is specificallyincorporated by reference as if fully set forth herein. It should benoted that whole blood under the same circumstances contains 0.23 ml O₂/ml. The FC-80 ScO₂ is linear from 760 to 200 Torr but declines quiterapidly below the lower level. The high oxygen diffusion coefficient(5.71×10⁻⁵ cm² /sec per second) indicates more than adequate FC-gas in aphysiologic sense. Similar studies concerning CO₂ solubility anddiffusion indicate that absorption and release are described by astraight line function. From these observations, metabolic tissue CO₂accumulations should theoretically be easily removed by fluorocarbonsolutions administered through a circulatory method.

Not only do fluorocarbons possess these unique physical gaseousproperties but they are for the most part non-toxic. The main acutetoxicity has been found to reside in free fluoride ion accumulationwhich occurs mainly from sonication. See, Clark et al., Fed. Proc. 34,pp. 1468-1477 (1979). The free ion can, however, be removed byrepetitive dialysis and the emulsion thereby rendered physiologicallyacceptable. Accordingly, the preferred embodiment nutrient liquid of thepresent invention, which has been dialysized and filtered through amillipore filter, has evidenced no toxicity either in short term or longterm use during circulation through cerebrospinal pathways of animals.One chief advantage of the CSF circulation route is that most or all thenutrient liquid can be removed by washing at the time of treatmenttermination. In this way long term cellular retention as previouslynoted for liver and reticuloendothelial cells in vascular circulationsof oxygenating liquids may be avoided.

In the preferred embodiment nutrient liquid of the present invention, anemulsification component is provided for permitting the emulsificationof the nutrient component with the oxygen transfer component of thatliquid. See Clark et al, Triangle II, pp. 115-122 (1972b); Clark et al,Microvasc. Res. 8, pp. 320-340 (1974). The best currently availablematerial for this purpose is believed to be block polymer polyols, whichare known to the art as "pluronics", of which, pluronic F68 has provento be a most efficient emulsifying agent. As used in a nutrient liquidas described more fully hereinafter, the toxicity from such a pluronicdetergent is negligible. At the present time, however, it is anticipatedthat other emulsification components which will permit the non-aqueoustransfer component of the nutrient liquid to become soluble with respectto the aqueous nutrient component of the nutrient liquid may be utilizedto provide solutions which have adequate physiologic perimeters. Suchother means of solubilizing fluorocarbons includes the formation ofmicelles, etc.

In the preparation of the preferred nutrient liquid, an important factorin producing an acceptable nutrient liquid is the achievement of anacceptable final osmotic pressure. The osmotic pressure of the nutrientliquid will depend upon the amount of the emulsification component, theparticle size of the fluorocarbon, and the ionic composition of theaqueous nutrient component. In accordance with the preferred method ofpreparing the nutrient liquid of the present invention, toxicemulsification components should be removed by dialysis. Fluorocarbonparticle size will be controlled by sonification time and filtering,while the ionic composition of the aqueous nutrient component will becarefully adjusted to produce a nutrient liquid possessing desiredosmotic characteristics. If desired, a final osmotic tuning may beaccomplished in accordance with the method of the present invention byadding ascorbic acid to the nutrient liquid.

In order to provide fully successful treatment of ischemic tissues, itis desirable to provide nutrient liquid for circulation around thosetissues which will compensate for relative or complete deficiencies ofblood transport metabolites. In addition to oxygen, other tissuemetabolic requirements include glucose, amino acids, ions, hormones,vitamins, etc. While in temporary treatment conditions, it may besuitable to temporarily omit one or more vitamin, hormone, ion, or aminoacid, for prolonged treatment and to produce the most desirable results,it is preferred to provide substantially all of the above mentionedmetabolites in the preferred nutrient liquid. It is at least desirableto provide in the nutrient liquid all components necessary to supportaerobic metabolism which will be available within the medium for use atcellular levels. Glucose deprivation of central nervous system tissuecauses a serious cellular metabolic deficiency, as does the same degreeof oxygen deficiency. Accordingly, by providing a total and finelyadjusted mixture that has all the necessary components for total cellsurvival, an extremely efficient and therapeutic liquid material isprovided which is ideal for circulation through the cerebrospinalpathways.

In order to illustrate the preferred method and composition of such anoxygen-nutrient material, the following example is provided.

EXAMPLE 1

Under conditions of replacing blood borne materials by perfusion allnutrients necessary for aerobic metabolism must be available within themedium for immediate use at cellular levels. As far as the centralnervous system is concerned, glucose deprivation causes as serious acellular metabolic deficiency as does the equivalency of oxygen lack. Toachieve the desired ends all known essential nutrients have been addedto the FC (fluorocarbon) emulsion. FC itself thereby serves the purposeof a gas transport system while the aqueous emulsion phase contains anarray of cellular metabolic essentials. The total and finally adjustedmixture has all the necessary ingredients for total cell survival. Thecombination material is referred to as an oxygen-nutrient formula(Ox-N), or oxygenated nutrient emulsion.

    ______________________________________                                        Method and Composition Preparation of Oxygen-Nutrient Material                ______________________________________                                        Reagents                                                                      1.  (A)    5% Commercial grade Pluronic F68 (Basic                                       Wynadotte).                                                            (B)    20% W/V FC-80 (3M Corporation)                                         (C)    Synthetic C.S.F.                                                   Sodium Chloride         7.3    gm/L                                           Potassium Chloride      300    mg/L                                           Calcium Chloride (dehyd)                                                                              200    mg/L                                           Magnesium Sulfate       300    mg/L                                           Sodium Phosphate (hepta)                                                                              200    mg/L                                           Sodium Bicarbonate      190    mg/L                                           Adjust the pH to between 7.380-7.420 with                                     10% Ascorbic Acid                                                             (D)    Bacitracin Inj. 50,000 U/vial (Pharmacy)                                      reconstitute with 10 ml saline to give a                                      concentration of 5000 U/ml. Use 0.2 ml for                                    each liter of perfusate to obtain a concen-                                   tration of 1,000 units per liter of perfu-                                    sate.                                                                  (E)    Essential Amino Acids (Pool) (Sigma) ,-                                           D-Glutamic Acid       11.8 mg                                      L-Glutamine                                                                          730.0 mg                                                               DL-Serine                                                                            26.3 mg                                                                D-Threonine                                                                          30.0 mg                                                                D-Lysine                                                                             38.8 mg                                                                D-Valine (optional)                                                                  19.0 mg                                                                D-Leucine                                                                            14.0 mg                                                                DL-Isoleucine                                                                        13.0 mg                                                                D-Phenylalanine                                                                      15.0 mg                                                                DL-Tyrosine                                                                          14.0 mg                                                                D-Methionine                                                                         4.5 mg                                                                        Before oxygenating the fluorocarbon emulsion                                  add 9.8* mg. amino acid and 200 mg dextrose                                   for each 100 ml of emulsion.                                           (F)    Steroid (Methylprednisolone sodium                                            succinate) 125 mgs. (The Upjohn Company).                                     Reconstitute the steroid with 2 ml of                                         diluent to obtain a concentration of                                          62.5 mg/ml. Add 0.5 ml of this mixture to                                     each liter of emulsion before oxygenation                                     (31.2 mg/L).                                                           (G)    1 N NaOH                                                               2. Materials                                                                  (A)    Sonifier Cell disrupter (Branson) Model                                       W185D                                                                  (B)    Waring Blender for mechanical dispension                                      of Pluronic Acid.                                                      (C)    Dialyzer tubing 7/8 in. (22 mm) (Thomas).                                     It is necessary to dialize the emulsion                                       to remove fluoride ions as well as other                                      low molecular weight contaminants.                                     (D)    Whatman Filter Paper #1 (46 × 57) (Thomas)                              The emulsion should be filtered to remove                                     particles originating from disrupted carbon                                   skeletons of flurocarbon during sonication.                            (E)    0.8 micron filter unit (Thomas). Sterili-                                     zation is accomplished by filtering the                                       emulsion through a micro filter.                                       (F)    CO.sub.2 tank (Welders Supply Company) CO.sub.2                               is used as a defoaming agent while                                            sonicating.                                                            (G)    100% O.sub.2 tank (Welders Supply Company)                                    CO.sub.2 is used as a defoaming agent while                                   sonicating                                                             (H)    100% O.sub.2 tank (Welders Supply Company) for                                saturating perfusate.                                                  (I)    Sterile Culture Flasks (Thomas) for                                           storing perfusate.                                                     (J)    Gas Dispersion Tubes (Fisher Scientific                                       Company) for equilibrating the emulsion                                       with O.sub.2.                                                          (K)    Aspiratory Bottle (Thomas)                                             a.       250 ml capacity-cut off 21/2" from                                            the neck with a glass cutter in order                                         to accommodate the macrotip for                                               sonification.                                                        b.       500 ml capacity -                                                             for equilibration of                                                          the emulsion with 100 ml                                                      capacity - 100% O.sub.2.                                             (L)    K50 Extension tubing. Capacity approxi-                                       mately 2.1 ml length 40.7 centimeters                                         (20 in.).                                                              (M)    Circulating Pump                                                       (N)    Sonification Assembly                                                  a.       Fill a container with crushed ice;                                            one that will allow drainage of the                                           water as the ice melts (a fish tank                                           will do).                                                            b.       On the serrated outlet near the bottom                                        of the aspiratory bottle connect                                              seven lengths (140 in.) of K50 extension                                      tubing. Place the bottle in the                                               ice bath and connect the tubing to                                            circulating pump.                                                    c.       Place the precooled Pluronic acid in                                          the aspirator bottle. Drape and                                               return extension tubing from the pump                                         over the side of the bottle. Drape                                            the tubes from the CO.sub.2 tank over the                                     side of the bottle and bubble slowly.                                         Carefully lower the macrotip into                                             the solution and start sonification.                                 3. Method 20% FC-80 (5% Pluronic (F68)) (w/v)                                 (A)    Place 25 gms of F68 + 250 ml of artificial                                    CSF in a Waring blender and blend at                                          a high speed for 2 minutes. The solution                                      will become very foamy. For best results                                      the solution should be refrigerated                                           overnight before using. This allows the                                       head of foam to settle and precools the                                       solution to the proper temperature for                                        sonification.                                                          (B)    Place the precooled Pluronic acid solution                                    in the aspirator bottle. Turn on                                              Sonifier. With a Pasteur pipette add                                          58.8 mls (100 gm) of FC-80 over a 30 minute                                   period sonifying throughout. Once added                                       allow the mixture to sonicate for 45                                          minutes. Be sure that the temperature                                         does not exceed 20° C.                                          (C)    Cut dialyzer tubing that has been pre-                                        soaked in artificial C.S.F. into 60-inch                                      strips. Fill each strip half full with                                        the mixture. Place strips in containers                                       filled with approximately 1000 ml, of                                         artificial C.S.F. Refrigerate and                                             allow to dialize for 48 hours. The                                            dialyzing solution should be changed                                          every twelve hours, and the emulsion                                          checked and transferred to additional                                         tubing since the volume is considerably                                       increased during dialysis.                                             (D)    After dialysis filter the solution through                                    Whatman #1 filter paper, then take the                                        total volume. 25 gm of Pluronic acid and 58                                   750 ml of emulsion. The former volume                                         represents 20% FC-80 and 5% F68 w/v ratio.                                    The emulsion should be kept in an ice                                         bath while processing.                                                 (E)    Add bacitracin to the emulsion. The pH                                        at this point should be between 6.5 and                                       6.8.                                                                   (F)    It is necessary to adjust the electrolytes                                    at this stage.                                                                Unadjusted electrolytes:                                                      Na = 127                                                                      K = 5                                                                         Cl = 126                                                                      CO.sub.2 = 1.5                                                                Osmolarity = 271                                                       It is necessary to add 696 mg NaCl/L of                                       emulsion in order to normalize the                                            electrolytes.                                                                 Adjusted electrolytes:                                                               Na = 131                                                                      K = 3.8                                                                       Cl = 130                                                                      CO.sub.2 = 3                                                                  Osmolarity = 303                                                       (G)    Using 1.0 N NaOH adjust the pH to between                                     7.380 and 7.420, then check the osmolarity                                    (Range 298-317)                                                        (H)    Sterilize the emulsion by filtering through                                   0.8 micron filter. The emulsion can be                                        frozen at -20° C. and is stable for several                            months.                                                                4. Immediately Before Using Emulsion                                          (A)    Add:    Glucose     0.8-2.5 gm/L                                                      Amino Acid  0.098 gm/L                                                        Steroid     31.2 mg/L (optional)                               (B)    Warm the emulsion to 37° C. and equilibrate                            with 100% O.sub.2 using a gas dispersion tube                                 for 30 minutes to obtain a pO.sub.2 of between                                580-660.                                                               (C)    A typical batch of FC-80 emulsion shows                                       the following properties:                                                     Na = 131 meq/L                                                                K = 3.8 meq/L                                                                 Cl = 130 meq/L                                                                CO.sub.2 = 3 meq/L                                                            Glucose = 186 mg. %                                                           Osmolarity = 311 mOsM                                                  (D)    A typical batch of oxygenated nutrient                                        emulsion contains:                                                            Fluorocarbon = 78.6 ml/L                                                      Pluronic Acid = 213 ml/L                                                      Na Cl = 7.3 gm/L                                                              Potassium Cl = 300 mg/L                                                       Calcium Cl = 200 mg/L                                                         (dehydrated)                                                                  Mg Sulfate = 300 mg/L                                                         Sodium Phosphate = 200 mg/L                                                   Sodium Bicarbonate = 190 mg/L                                                 Amino Acid Pool = 0.098 gm/L                                                  (added to fluorocarbon)                                                       Manitol Injection = 50 ml/L                                                   USP 259                                                                       Bacitracin = 5000 units/L                                                     Gentamicin = 80 mg/L                                                          Dextrose = 2 gm/L                                                             Ascorbic Acid = 0.5 ml/L                                                      (10%)                                                                         Sterile Water = remainder per liter                                    ______________________________________                                        Gas Characteristics After Oxygen Equilibration                                             Unsaturated                                                                              Saturated                                             ______________________________________                                        pH           7.231      7.342                                                 pCO.sub.2    3.7        5.7                                                   pO.sub.2     190        640.5                                                 ______________________________________                                         *Siegel et al., Basic Neurochemistry (2nd edition), Little, Brown and         Company, Boston p. 297.                                                  

In order to provide an indication of the efficacy of the preferredtreatment methods, the following examples are provided:

EXAMPLE 2

For reasons of simplicity and reproducability a model continually in usein applicant's laboratory has been employed. Osterholm, J. L.,Pathophysiology of Spinal Cord Injury, C. C. Thomas, Springfield, Ill.(1978). Extensive experience with spinal cord injury in terms ofstandardization, quantitative histological studies, regional blood flowand biochemical parameters suggested these procedures. A primarypathophysiologic event in that model has been determined to be discreteregional ischemia. A microcirculatory flow failure within the injuredregion has been documented by many study techniques includingmicroangiography, distribution of intravascular particulate materials,hydrogen-platinum flow studies, regional istopic techniques and lactateaccumulation. Recent C 14 antipyrine microregional blood flow studiesconducted in applicant's laboratory have accurately delineated themagnitude of ischemia in the injured cord. Within one hour the regionalgrey matter flow drops from the control of 44 cc/100 gm/min to only 2cc/100 gm/min. The white matter is also ischemic. Blood flows in theseregions are depressed from 15 cc/100 gm/min to 1-2 cc/100 gm/min.

From these observations, standarized spinal cord injury causes arestricted ischemic lesion which can be easily studied and quantitated.In this rigid system therapeutic treatment effects are readily detectedby comparison with our extensive untreated injury data. It should benoted here that the mechanical injury forces used in these experimentsare substantially above saturation and all wounded animals are renderedpermanently paraplegic.

Circulation Experiments

Experiments were carried out by continuously injecting either saline orOx-N emulsion saturated with O₂ at 1 atm into the distal subarachnoidspinal space. The outflow (withdrawal) of the diagnostic fluid was atthe cisterna magna. Infusions were begun immediately after severewounding. An infusion rate of 3 ml/minute was easily achieved, and thisrate was maintained for two hours.

Oxygen

Prior to lumbar spinal infusion we were able to develop pO₂ tensions of535±89 mm O₂ in the Ox-N emulsion by simply bubbling 100% oxygen throughthe solution. Upon exit at the cisterna magna after traversing theentire spinal subarachnoid space the pO₂ had fallen to 243±63. Theoxygen difference between entering and exit was 292±63, or a 55%decline, which is statistically significant at the P<0.001 level. Thisfinding indicates a rapid pO₂ exchange during the thirty seconds or lesstransit time. For various technical reasons our initial pO₂ was lowerthan can be achieved under idealized circumstances. More recently it hasbeen possible to regularly attain pO₂ of about 650 Torr. Even betterexperimental results might have now been obtained under conditions ofhigher O₂ tension.

Carbon Dioxide

FC-80 is an efficient CO₂ exchange and transport agent, and the emulsiontherefore easily extracts tissue CO₂. This is indicated by an initialemulsion pCO₂ of 2.7 Torr which rose to 16.0 Torr after the tissueperfusion contact. This represents a 593% increase in FC-80 CO₂(P<0.001). The emulsion also removes other acid metabolites since insome experiments the inherent buffering capacities were exceeded as theexit fluid pH exhibited a considerable depression toward the acid side(original pH 7.4, exit pH 7.0). This pH change exceeded any acidcontribution by the collected CO₂, and amounted to 0.248 molelactate/hour.

A. Cross Sectional Area (Edema)

Frozen tissues were sectioned and stained (H & E, and acid phosphatase).The sections were evaluated by projection to 25× magnification andpreselected lesion parameters measured by means of a compensating polarplanimeter. There was considerable increase in the untreated injury cordcross sectional area (1280 mm²) which was significantly reduced in theOx-N experiments, (896 mm²). We have assumed that this substantial crosssectional cord area increase is caused by edema fluid. In the course ofother experiments, the degree of edema appearance has been quantified.It was found that net water accumulation at those post injury timesranged from 25% to 40%. The absolute reduction in cross sectional areaby the Ox-N treatment is significant at the P=0.001 level.

Lesion Size

Using our standard sampling methodology which includes skip serialsections throughout the injury region, and analysis by quantificationtechniques, the degree of injury induced hemorrhagic necrosis can bedetermined. With the perfected injury system the lesion size at any timepoint can be reliably predicted. The effects of saline and Ox-Ncirculations upon lesion size were compared to each other and to ourestablished untreated values. The results are summarized in Table I:

                  TABLE I                                                         ______________________________________                                        LESION SIZE                                                                   2 Hour Injuries                                                               % Grey         % White      % Total                                           ______________________________________                                        Stan- 79.5 ± 16% SD                                                                            30.1 ± 9% SD                                                                           39.5 ± 10% SD                              dard                                                                          Injury                                                                        (No                                                                           Infu-                                                                         sion)                                                                         Saline                                                                              78.3 ± 15% SD                                                                            25.0 ± 14% SD                                                                          34.4 ± 12% SD                              Circu-                                                                        lation                                                                        Ox--N 47.4* ± 17% SD                                                                          12.8* ± 2% SD                                                                           19.2* ± 10% SD                             Circu-                                                                        lation                                                                        ______________________________________                                         Table I                                                                       Percentages are expressed in terms of total tissue area lesioned by           hemmorrhagic necrosis for grey, white or total cord area two hours after      severe injury with the various treatments.                                    (*Statistical Significance P = <0.01. The saline values are not               significant).                                                            

The data indicates a highly significant degree of protection againstinjury lesions afforded by the Ox-N circulation treatments. The actuallesions are halved by the treatment and this remarkable stabilizingeffect upon the important white matter tracts would be anticipated tosubstantially improve the final functional result attending severespinal cord injury.

Anterior Horn Cells

A technique of counting the anterior horn cells which contain visibleacid phosphatase histomchemical reaction product has been developed inthis laboratory. The procedure has been previously used to assessischemic cellular effects in terms of cellular survival and/or lysistime.

From Table II it can be seen that untreated injury has a highly lethaleffect upon anterior horn neurones. Within the two hour experimentaltime period, more than 97% of all cells at the injury center undergocytoplasic lysis. Ox-N infusions stabilized the injured cells as 60% ofall neurones were protected from lysis.

                  TABLE II                                                        ______________________________________                                        ANTERIOR HORN CELLS                                                           ______________________________________                                        Control               34 ± 2 (SD)                                          Injury                2 ± 1.73*                                            Injury + Ox-N        21 ± 5.12**                                           circulation                                                                   ______________________________________                                         Table II                                                                      Number of anterior horn cells containing acid phosphatase reaction produc     within well defined cytoplasmic borders, (*statistical difference from        control P <0.001,                                                             *Difference from injury alone P <0.001).                                 

Spinal Cord Adenosine Triphosphate (ATP)

Biochemical ATP tissue determinations were undertaken to determine themetabolic oxidative state of injured spinal tissues. This metabolite wasselected for study since it reflects the progress of normal oxidativemetabolism. ATP levels fall very rapidly under sufficienthypoxic-ischemic conditions. Untreated injured cords have a 200% ATPdecline in one minute. In the current experiments ATP levels would beexpected to reflect (1) the cellular oxidative capability and (2)functional cellular viability. The latter aspect is especially importantin terms of cellular integrity which was discussed in the preceedingsection.

From Table III it can be seen that 2 hour injury causes a four and threefold drop in grey matter and white matter ATP respectively. Thisinformation amply supports other observations about the degree ofregional cord tissue ischemia after impaction. ATP was found insignificantly higher concentration in the Ox-N experiments than notedafter saline circulation alone. The high energy compound suffered only a30% fall from normal in the oxygenated perfusion group which contrastsvividly with the 300-400% loss found with the saline treatments.

                  TABLE III                                                       ______________________________________                                        ATP LEVELS (μmol/gm) (2 hours post injury)                                        Injury & Saline                                                                           Injury & Ox-N                                                                             Control                                        ______________________________________                                        Grey Matter                                                                            0.46          1.24*       1.88                                       White Matter                                                                           0.40          0.87*       1.23                                       ______________________________________                                         Table III                                                                     ATP tissue levels in control, saline and OxN injured cords. The differenc     between saline and OxN is significant *(P =0.05). Although not shown in       the Table, the OxN treatments also statistically increase ATP in spinal       cord regions directly above (P < 0.001) the injury site.?                

Comparison of the above results to those later reported by R. E.Hanseabout, R. H. C. Van Der Jagt, S. S. Sohal, and J. R. Little,Journal of Neurosurgery 55, pp. 725-732 (1981) is of interest.Hanseabout et al report the use of a commercial oxygenated fluorocarbonartificial blood perfusate to treat experimental spinal cord injuries.Treated dogs are reported as showing improved motor function morerapidly and as having a better final hind limb functional result thandid controls. To some extent, this non-prior art report confirms thespinal cord injury findings reported here.

EXAMPLE 3 Cerebrovascular Ischemia

Initial studies have been conducted to determine the efficiency of Ox-Nemulsions in protecting the brain against profound ischemia. We employedthe cat brain and utilized right hemispheric regional vascularinterruption so that the left cerebral hemisphere might serve as aninternal control. The middle cerebral artery of cat is accessiblethrough the bony orbit. It lies immediately above the optic nerve afterthe canal has been opened and can be identified with certainty in thatposition. Preliminary experiments determined that an inconstant cerebralfield was devascularized by occluding the middle cerebral artery. Itbecame apparent that collateral blood flow via the anterior andposterior cerebral arteries supplied some retrograde filling into theexperimental region. This phenomenon could be largely prevented byconcommittantly reducing the mean systemic blood pressure to 70 mmHg byexternal bleeding. Hemorrhagic hypotension plus middle cerebral arteryocculsion yielded a reasonably constant ischemic cerebral lesion fromanimal to animal.

In that model either saline or Ox-N were circulated from the rightcerebral ventricle to the cisterna magna at a rate of 3 ml/min. Cerebraltissues were harvested one hour after vascular occlusion by immediateimmersion in liquid Freon. The tissues were sectioned in the frozenstate and reacted with luciferin upon photographic film. A combinationof high energy cellular metabolites plus luciferin react to emit visiblelight, which is recorded upon the film. Tissues removed from salinetreated ischemic cerebral regions were uniquely devoid ofphospholuminescence, while the opposite hemisphere demonstrated thisreaction to a degree similiar to that found in normal animals. Middlecerebral ischemic tissue samples from Ox-N treated animals containedsufficient high energy materials to demonstrate a positive histochemicalhigh energy reaction one hour after vascular arrest.

EXAMPLE 4 Profound Spinal Cord Ischemia

The combined evidence from spinal cord injury and middle cerebral arteryocclusion models demonstrate that the preferred oxygenated nutrientemulsion can be circulated to maintain cellular integrity and aerobicmetabolism under the stress of profound regional ischemia. A third modelwas utilized to determine if vascular deprived neurones perfused viacerebrospinal fluid pathways with oxygenated-nutrient would continue toperform a physiologic function. A transthoracic aortic ligation justdistal to the left subclavian effectively devascularizes the cervical,thoracic and lumbar cat spinal cord. In some examples the lower brainstem was also found ischemic by regional flow studies. The mid and lowerthoracic cord are universally and profoundly blood deprived by thisvascular interruption. Animals under light Ketamine anesthesia weretreated by circulating from the lumbar subarachnoid space to thecisterna magna with either saline or Ox-N solutions. Respiratorymovements were evaluated in these experiments. The lungs were ventilatedby positive presence respiration, but the mechanical movements areeasily distinguished from neuromuscular respiratory contractions. Thisis especially so since for the most part the respiration andneuromuscular drive occur at separate times and are largelyasynchronous. Following the aorta ligation all physiologic neuromuscularrespiratory movements progressively diminished to total cessation after5-10 minutes in the saline treated cats. The arrest obtains forintercostal muscles as well as diaphragmatic contractions. The Ox-Ntreated animals, on the other hand, continue to respire in anessentially normal neuromuscular sequence. The respiration, under thoseconditions, were often of irregular rates, diminished in amplitude, andshowed some individual magnitude variations. The singular differencebetween saline and Ox-N circulations is the universal persistence ofrespiration in the latter group. It is also true that Ox-N sustainedsufficient chest bellow movements so that if the chest were closed therespirations were clinically adequate to support life.

EXAMPLE 5

Experiments have also been conducted to determine the efficacy of theherein disclosed methods on global cerebral ischemia induced in cats.

Although the Ox-N emulsions of the present invention are oxygenatable bybubbling gas through them, perfusate from stroke animals were initiallyfound to have oxygen pressures (pO₂) below those known efficient oxygenexchange values (pO₂ less than 200) for the fluorocarbon component ofthe material. See Navari et al, supra. Accordingly, the pump oxygenationsystem described above in connection with FIG. 13 was developed tooptimize fluorocarbon O₂ saturation. As mentioned above, this systemcomprises a heat exchange-oxygenator which was coupled to recirculating,warming and delivery pumps. This system rapidly oxygenates the emulsion(pO₂ =645[mean] Torr) at 37° C. with oxygen gas delivered at 7 L/min.

Global cerebral ischemia experiments were conducted on cats afterbrevital induction and nitrous oxide oxygen (70-30%) anesthesia. Adouble lumen inflow cannula of the type described above wassterotactically placed into a lateral cerebral ventricle while an exitcannula was inserted either into the cisterna magna or lumbar theca.When the conduits are properly installed, the CSF pathways have littleresistance and a mean flow perfursion rate of 6.0 cc/min. can beachieved through the animals without intracranial pressure alterations.Entry and exit fluid were collected for metabolic studies. Both gaseswere normalized by respiratory adjustment. Further experimentalmanipulations awaited electroencephalograph (EEG) normalization.Cerebral ischemia was produced by the combined insult of hemorrhagichypotension (mean arterial blood pressure lowered to 30±3 mm Hg) plussimultaneous carotid artery clamping. This method caused a bihemisphericisoelectric EEG within 5-8 minutes. After sustained and total cerebralelectro silience for 15 minutes, the carotid arteries were unclamped andthe withdrawn blood reinfused.

A well accepted measure of cerebral function, the EEG, was used toassess both the degree of insult and subsequent discovery. A computerbased EEG method, compressed spectral analysis, was used to determinebrain activity. A Nicolet Instrument Corporation "MED-80" computerutilizing frequency analysis package "Super C" was used with thefollowing setup parameters:

2 channels, 1024 SEC. EPOCH, 1024/PTS.EPOCH

2 sweep average/printout.

The total output is expressed in (microvolts²) assuming a constantsource impedence of 1 ohm. The data presented here is the total cerebralpower 0.3-25 Hz in picowatts. Recordings were made from skull electrodesat maximum sensitivity of 1 picowatt. Since a steady state prestroke EEGwas obtained, each animal served as its own control.

Ten animals had cannulas placed and the stroke accomplished withoutperfusion. A second control group of ten animals were treated similarly,but were also perfused through the ventriculo spinal (lumbar) route withnutrient solution without fluorocarbon. There were no apparentdifferences found for post-stroke electroencephalographic activity inthese groups. As a measure of stroke severity, 13 animals (of 20) hadpersisting electrocerebral silience. Of the remaining animals, 5 gainedonly 2% of their base line power while two had 10% power return withinthe 4 hour experimental period. FIG. 4 is a representative EEG powertracing from the left and right cerebral hemispheres of a cat perfusedonly with nutrient solution without fluorocarbon and which exhibitedpersisting electro-cerebral silience during the 4 hour experimentalperiod. The tracings are read from bottom upwards. Normal activity isseen in the lowest tracing and is totally arrested by the ischemicinsult half way through the first grouping. There is electro-cerebralsilience thereafter througout the experimental period.

Thirteen cats underwent the same experimental procedure, but wereperfused immediately after ischemia with bubble oxygenated nutrientsolution (pO₂ =400). For these cats, the flow rate was 4 ml/min withwithdrawal from the umbar theca. Five exhibited continuedelectro-silience whereas 8 demonstrated EEG recovery from 5% (6 animals)up to 34% (2 animals). FIG. 5 is a representative EEG tracing of one ofthe eight animals demonstrating 5% recovery after perfusion withoxygenated nutrient emulsion (pO₂ =400).

A fourth group of 7 cats was perfused with pump oxygenated nutrientsolution (pO₂ =645) at 6 ml/min. with withdrawal from the cisternamagna. All cats in this group regained some electrocerebral activity.The final total power which returned ranged from 5 to 88% of theprestroke base line (average 22%; p<0.01 compared to all non-oxygengroups). The electroencephalographic activity recovered generallythroughout the 4 hour recovery period with the returning total cerebralpower exhibiting a first order relationship as a function of time. Atthe observed recovery rate all animals should achieve completely normalEEG power spectra within 8 hours. An oxygen dependent EEG response isseen when non-oxygenated, bubble oxygenated (pO₂ =400), and pumpoxygenated (pO₂ =645) groups are compared as electrocerebral activityrecovery greater than 5% was found in 10%, 62% and 100% respectively.FIG. 6 is an EEG tracing of the animal showing 88% return ofelectrocerebral activity within 4 hours after perfusion with oxygenatednutrient emulsion (pO₂ =645). The asymmetry between hemispheres is anindividual variation for this animal.

FIG. 7 is a portion of an EEG tracing showing the recorded effect onelectro-cerbral activity of a temporary perfusion failure. This animal,which was perfused using the pump-oxygenated (pO₂ =645) nutrientemulsion described above, experienced an interruption (pt. A) inperfusion for a time period of approximately 1 hour, whereupon perfusionwas resumed (pt. B). As seen in this tracing a major deterioration ofEEG activity occurred following cessation of perfusion, and resumedthereafter, confirming that the present method in fact sustains EEGactivity.

In FIG. 8, the effect of a diminished perfusion flow rate of oxygenatednutrient emulsion is shown on the rate of glucose metabolism, andlactate and pyruvate concentration. In accordance with theabove-described ventriculo-lumbar perfusion procedure using bubbledoxygenated (pO₂ =400) nutrient emulsion, flow rate with nutrientemulsion without fluorocarbon was established at about 5.0 ml/min. Abase line cerebral metabolic rate of glucose metabolism (CMRGl) wasestablished prior to stroke, which was followed after 15 minutes withthe perfusion of the oxygenated nutrient emulsion. CMRGl, which hasrecovered somewhat after 1 hour, is seen to decline rapidly as the flowrate of perfusate declines. Similarly, lactate levels riseprecipitiously with flow rate decay. These results once again confirmthat the flow of oxygenated nutrient emulsion through the cerebralspinal pathway should be maintained at acceptable rates in order tosustain neurologic tissue.

In FIG. 9, the mean recovery percent for the four groups of animalsdiscussed above is presented in the form of a bar graph. It is presentlypreferred to insure that the pO₂ value of oxygenated nutrient emulsionupon input is great enough to insure that efficient oxygen transfercapabilities are maintained at the selected flow rate. For the FC lumbargroup, exposure of oxygenated nutrient solution to certain tissueregions when its oxygen exchange value was below the known efficientoxygen exchange value (pO₂ less than 200) for the fluorocarbon componentof this material may have occurred. This may be true even though themean oxygen exchange value of the withdrawn emulsion is above 200.Accordingly, it is presently preferred to maintain the pO₂ value ofwithdrawn oxygenated nutrient emulsion at twice this minimum, or atabove 400, either by raising the input pO₂ value to much higher levels,as with the ventriculo-cisternal animals described above, or byincreasing the flow rate of oxygenated nutrient emulsion through theanimal to maintain those values. In smaller animals, such as cats, thesize of the cerebro spinal pathways creates hydraulic resistance whichlimits the flow rates which may be achieved at atmospheric pressuresusing certain pathways. In such animals, higher oxygen exchange valuesand shorter perfusion routes, such as the ventriculo-cisternal perfusionroute, are preferred. In larger animals, such as humans, it is notanticipated that flow rates will be so limited. Nonetheless, high pO₂values (at least 50% preferably 80+% of the maximum obtainable pO₂) arepreferred to minimize the volume of perfusate necessary to perform agiven treatment and to provide an additional margin of safety at theselected flow rate.

Samples of the perfusing fluids for the animals of this example wereremoved at predetermined times from entry and exit perfusion ports foranalysis of lactate and pyruvate under a single blind condition. Theresults are summarzied in Table IV:

                                      TABLE IV                                    __________________________________________________________________________    Levels of lactate and pyruvate in cerebral spinal fluid perfusate before      (baseline), during (isoelectric) and following (reflow) global ischemia       in cats.                                                                      Data are expressed in mg per 100 ml of perfusate and the values are           means ± standard error. Six animals were perfused with NS.sup.1 and 7      with                                                                          OFNS.sup.2 solution. After collecting the perfusate in tubes a 4#C, the       samples                                                                       were stored at -80° C. for analysis. Lactate and Pyruvate were         assayed by a                                                                  Sigma Method (Sigma Technical Bulletin #726, Oct. 1968 and #862, Oct.         1969)                                                                         and conducted by Jefferson University Clinical Laboratories.                  __________________________________________________________________________                                  Lactate/                                        Experimental                  Pyruvate                                        Period  Lactate     Pyruvate  Ratio                                           __________________________________________________________________________    Baseline.sup.+                                                                        3.6 ± 1.1*                                                                             0.5 ± 0.1                                                                             7.2                                            Isoelectric.sup.+                                                                     8.1 ± 1.9*                                                                             0.5 ± 0.1                                                                            16.2                                            __________________________________________________________________________             NS   OFNS  NS   OFNS NS OFNS                                         __________________________________________________________________________    Reflow (5 min)                                                                        21.9 ± 11                                                                        10.0 ± 1.0                                                                       0.5 ± 0.1                                                                       1.2 ± 0.6                                                                       43.8                                                                             8.3.sup.+                                    Reflow (4 hr)                                                                          8.9 ±  3.3                                                                      10.4 ± 3.8                                                                       0.5 ± 0.1                                                                       1.7 ± 0.7                                                                       17.8                                                                             6.1.sup.                                     __________________________________________________________________________     *During baseline and isoelectric time periods all cats were perfused with     NS.                                                                           .sup.+ p < 0.01 when compared to baseline lactate. p < 0.025 when compare     to the ratio of reflow (5 min) perfused with NS.                              .sup.1 As used herein, NS refers to the nutrient solution of Example 1        without fluorocarbon component.                                               .sup.2 As used herein, OFNS refers to the preferred oxygenated,               fluorocarbon nutrient emulsion of Example 1.                             

In animals perfused with nutrient solution without fluorocarbon theconcentration of lactate during the actual stroke (isoelectro) was ofthe normal CSF value. The lactate level rose percipitiously, anadditional 440%, within 5 minutes of restoring the blood pressure andblood flow through the carotid arteries. Thereafter the level declinedduring the 4 hour period to 147% of base line. In contrast to thelactate data, the pyruvate concentration remained constant through theperfusion period.

When animals were perfused with oxygenated nutrient emulsion, on theother hand, the percipitious increase in lactate did not occur; insteadthere was a modest 52% rise during the initial 5 minute period, and thelevel thereafter remained stable. Significantly, in the oxygenatedseries the concentration of pyruvate more than doubled during theinitial 5 minutes and continued to increase gradually during theremainder of the 4 hour period. The net production of lactate andpyruvate are often used as indictators of anarobic and aerobicglycolysis, respectively. Since these compounds change under differentcircumstances the expression of lactate/pyruvate (L/P) ratio bestillustrates the net metabolic effects. A high L/P ratio indicates thatanarobic glycolysis predominates. It is common practice, therefore, touse the L/P ratio as a sensitive indicator of the redox state of cells.Perfusion oxygenation in accordance with the present inventionssignificantly (p≦0.01) lowered the L/P ratio when compared tonon-oxygenation (8.3 vs. 43.8). It is further evident that theoxygenated 4 hour L/P ratio is additionally lowered, whereas thenon-oxygenated values are still 5 times greater than the control.

Although the oxygenated nutrient perfusate transit time through thebrain is only a few seconds, significant oxygen extraction does occur.It was determined by the pO₂ difference between inflow and outflowfluids that oxygenated nutrient emulsion lost pO₂ =210 (mean) during itsintracerebral passage. Also unique to the oxygenated nutrient emulsionstudies was a rising carbon dioxide presence in the exit fluid which didnot occur in non-oxygenated experiments. The pCO₂ rose 5 fold in thesefluids over the four hour period (pCO₂ =6.0 vs. 3.0). It is consideredthat the appearance of carbon dioxide is important since it is a normalproduct of aerobic metabolism.

In FIGS. 10 and 11 levels of potassium in perfusate before (base line),during (isoelectric) and following (reflow) global cerebral ischemia incats are represented. Data are expressed in micro equivalents perminute, and the values are means ± standard error. Five animals wereperfused with nutrient emulsion without fluorocarbon, and six withoxygenated fluorocarbon emulsion. After collecting the perfusate intubes at 4° C., the samples were stored at -80° C. for analysis.Potassiums were assayed by atomic absorption spectrophotometry. Duringthe base line and isoelectric time periods, all 11 cats were perfusedwith nutrient emulsion without fluorocarbon. There were no significantdifferences between isoelectric and base line levels of potassium in theperfusate. As seen from FIGS. 10 and 11, significant differences in thevalues of potassium were observed beginning almost immediately withperfusion (at time 0) and extending throughout the 4 hour experimentalperiod.

FIG. 12 discloses the effects on glucose metabolism forventriculo-cisterna perfused animals subjected to the stroke andreperfusion procedure of this example. In accordance with the inventionof Dr. John Lewis Alderman, one of these animals was perfused with twicethe glucose concentration (372 mg %) of that used for the remaininganimals described herein. As seen from FIG. 12, the glucose metabolismof animals provided with oxygenated nutrient emulsion (glucose =186 mg%) is generally superior following reperfusion to the metabolism rate ofthe control receiving that solution without fluorocarbon. In view of thesubstantial increase in glucose metabolism exhibited by the animalhaving a "double glucose" solution (372 mg %), it is presently preferredto include at least such elevated glucose concentrations in perfusionsperformed in accordance with the method of the present invention.

These experimental results demonstrate that extravascular perfusion ofoxygenated nutrient emulsion affects a significant reversal of theadverse cerebral metabolic effects induced by the experimental strokecondition. Coincident with the improve metabolic state electrocerebralactivity returned. These findings indicate that extravascularly suppliedoxygen, glucose and other nutrients were taken up and metabolized inamounts sufficient to restore high energy compounds and therebyreactivate membrane ionic pumps and reinstitute electrocerebralactivity.

Oxygenated-fluorocarbon-nutrient-emulsion caused no detrimental effectson vital physiologic functions such as heart rate, blood pressure orelectrocerebral (EEG) activity when perfused through the ventricularsystem for four hours of cats not subjected to the stroke paradigm.These animals exhibited no ill effects after 5-8 months, and were killedfor a double blind neuropathologic examination of the brain, spinal cordand subarachnoid spaces. No gross or microscopic changes were observedand the specimens were indistinguishable from non-perfused animals.

In view of the above, those of ordinary skill in the art will recognizethat various modifications can be made to the methods and apparatusdescribed above without departing from the scope of the presentinvention. For example, it should be understood that, the injection andwithdrawal catheters used to perform the herein described method shouldbe sealed with respect to the skull so that a water and bacteria tightseal is created between these catheter and skull. Although conventionalbone wax has been used for creating this seal in the feline experimentsdescribed above, fitting 22(a) preferably comprises a double threadedsleeve which is threaded into a bone aperture, and in turn receivescomplimental threads formed on injection catheter 20a. Such attachmentmeans, particularly when used with a ventricular injection catheter,should eliminate any need for total head immobilization during humantreatment.

It should also be understood that the oxygenated nutrient emulsions ofthe present invention may contain various therapeutic agents includingfree fatty acids, prostaglandins, prostacyclins, cyclic nucleotides andhormones.

As seen from the above, it is desired to maintain the the pO₂ level inthe withdrawn fluid at levels which are substantially above the minimumlevel of efficient oxygen exchange of the subject fluorocarbon. For thefluorocarbon nutrient emulsion described above, that minimum(unsaturated condition) occurs at a pO₂ equal to about 190, which isabout 30% of the readily achieved maximum pO₂ level. (pO₂ =645) Asdescribed above, it is preferred to perform the treatment method of thisinvention so as to maintain the pO₂ of the withdrawn oxygenated nutrientemulsion at a pO₂ above 400, that is, at a pO₂ level which is abouttwice the minimum level of efficient oxygen exchange for the subjectfluorocarbon. It is presently anticipated that a similar differentialshould be maintained in practicing the present invention utilizingoxygenated nutrient emulsions having other oxygenatable componentsexhibiting different ranges of efficient oxygen exchange.

The methodology described requires the formulation of a physiochemicalfluid which must be adequately oxygenated, temperature controlled anddelivered under well controlled conditions. The perfusion system of thepresent invention may be routinely placed by trained animal surgeons.Neurosurgeons commonly possess skills necessary to implant treatmentports in accordance with the present invention in humans. The procedureis relatively simple and can be quickly accomplished with availableinstruments. The oxygenated nutrient emulsion treatment delivery systemof the present invention has certain similarities to the arterialheart-lung machine. Major differences, however, include the use of acomplex synthetic fluid for cerebral spinal perfusion, the routeperformed by cerebral spinal perfusion is an extravascular one, andthere is no known limitation on perfusion time in accordance with theherein disclosed method. Oxygenated fluorocarbon nutrient emulsiontolerates pumping mechanics well and the exit fluid can either bediscarded or recirculated. Formed blood elements, on the other hand, arefragile and lyse under prolonged recirculation conditions. It ispresently contemplated that cerebral-spinal fluid perfusion support willneed to be carried out until the vascular system can once again takeover. Surgical revascularization or bypass procedures will in some casesbe necessary to accomplish this end. The return of cerebral vascularcompentency can be assessed by measurements of regional blood flow,electro cerebral activity, and the metabolic configuration of the exitperfusion fluid. One foreseeable complication of this technique isbacterial infection, and rigorous attention to ambient sterility,millipore filtering, and antibiotics should reduce this hazard toacceptable levels. Safeguards have been built into the pumping system toimmediately stop delivery if either the inlet or outlet becomeobstructed.

CONCLUSION

As seen from the above examples, and the foregoing description,circulation of the preferred embodiment nutrient liquid is capable ofsustaining cellular integrity, aerobic metabolism and ongoing neuronalfunction. Even for neurons deep within the spinal cord (grey matter) theprocess has been successful in nurturing the ischemic neurons. Theability to sustain the central nervous system in a lethally ischemicfield which persists for longer than a few minutes has never beenaccomplished before. The extravascular pathway has not been employed asa global nutrient route prior to the present invention, nor has thecombined use of oxygen rich emulsion which also contains the otherdisclosed novel components been know to the art.

As seen from the above experiments, the methods, compositions and systemof the present invention are capable of providing substantial amounts ofoxygen to neurologic tissues to be treated, while at the same time,removing the by-products of aerobic metabolism, including carbondioxide, which have been found to exist in substantially higherconcentrations in the exit, diagnostic fluid. Similarly, as discussedabove, rapid, normally lethal, lyses of anterior horn cells is readilypreventable through the treatment of the present invention, protectingat least 60% of the cells through this modality. Similarly, high energyphosphate metabolism utilizing both oxygen and glucose is maintained atsubstantial levels. Accordingly, the methodology of the presentinvention represents a substantial advance in the treatment of centralnervous system tissue. Prior to this invention there was not a methodavailable to sustain central nervous tissues after a few minutes ofprofound ischemic insult. This invention should revolutionize thetherapeutic capabilities by providing therapeutic approaches for stroke,aneurysm, brain injury, vasospasm, senility, tumors, coma, spinal cordinjury, ischemia, post shock, post cardiac arrest and central nervoussystem poisoning.

It is further anticipated that the treatment method of the presentinvention should make it possible to interrupt the cerebral blood supplywith some impunity for surgical maneuvers not heretofore possiblewithout great attendant risk of producing cerebral infarction. Those ofordinary skill in the art will recognize that future development mayresult in perfection of the oxygenated nutrient emulsion composition,delivery rates, treatment times, the width of the therapeutic window inwhich treatment may be instituted and the correlation of behavioralfunctions in surviving animals with normalization of cerebral chemistryand electrographic activity. Nonetheless, by any standard, the presentinvention provides a dramatic, yet clinically acceptable, therapeuticmethod for treating ischemic neurologic tissue.

What is claimed is:
 1. A method of treating hypoxic-ischemic neurologictissue, in mammals, comprising:(a) providing a physiologicallyacceptable oxygenated fluorocarbon emulsion capable of transferringoxygen to hypoxic-ischemic neurologic tissue contacted thereby; (b)injecting a substantially continuous stream of said fluid into thecerebrospinal fluid pathway at a first injection point; (c)substantially continuously withdrawing fluid from said cerebrospinalfluid pathway at a second point which is selected to create acirculation of said oxygenated fluorocarbon emulsion in the vicinity ofsaid hypoxic-ischemic tissue, said circulation being sufficient tosupport significant aerobic respiration of a portion of saidhypoxic-ischemic tissue;whereby hypoxic-ischemic neurologic tissue willbe oxygenated by said fluid.
 2. The method of claim 1 wherein said firstpoint is within a cerebral ventricle.
 3. The method of claim 2 whereinsaid first point is within the lateral ventricle.
 4. The method of claim1 wherein said second point is within a subarachnoid space.
 5. Theinvention of claim 4 wherein said subarachnoid space is a spinalsubarachnoid space.
 6. The invention of claim 5 wherein said spinalsubarachnoid space is a lumbar spinal subarachnoid space.
 7. The methodof claim 1 wherein said second point is within a cerebral cistern. 8.The invention of claim 5 wherein said cerebral cistern is the cisternamagna.
 9. The method of claim 1 wherein said fluid is injected at a rateof between 1 ml/minute and 200 ml/minute.
 10. The method of claim 9wherein said fluid is withdrawn at a rate which selected to maintain thehydraulic pressure of said fluid with the CSF pathway at between about25 mm and 250 mm of water.
 11. The method of claim 1 wherein said fluidis injected at a rate of between about 20 ml/min/100 gms ofischemic-hypoxic neurologic tissue to be treated and 60 ml/min/100 gmsof said tissue to be treated.
 12. The method of claim 11 wherein saidinjection rate is about 45-60 ml/min/100 gms of tissue to be treated.13. The method of claim 1 wherein said fluid is injected at a hydraulicpressure which does not exceed 250 mm of water.
 14. The invention ofclaim 1 wherein said first point is within a subarachnoid space.